First report of KPC variants conferring ceftazidime-avibactam resistance in Colombia: introducing KPC-197

ABSTRACT Resistance to ceftazidime-avibactam (CZA) due to Klebsiella pneumoniae carbapenemase (KPC) variants is increasing worldwide. We characterized two CZA-resistant clinical Klebsiella pneumoniae strains by antimicrobial susceptibility test, conjugation assays, and WGS. Isolates belonged to ST258 and ST45, and produced a KPC-31 and a novel variant KPC-197, respectively. The novel KPC variant presents a deletion of two amino acids on the Ω-loop (del_168–169_EL) and an insertion of two amino acids in position 274 (Ins_274_DS). Continued surveillance of KPC variants conferring CZA resistance in Colombia is warranted. IMPORTANCE Latin America and the Caribbean is an endemic region for carbapenemases. Increasingly high rates of Klebsiella pneumoniae carbapenemase (KPC) have established ceftazidime-avibactam (CZA) as an essential antimicrobial for the treatment of infections due to MDR Gram-negative pathogens. Although other countries in the region have reported the emergence of CZA-resistant KPC variants, this is the first description of such enzymes in Colombia. This finding warrants active surveillance, as dissemination of these variants could have devastating public health consequences.

(bioMerieux, Marcy l'Etoile, France).As shown in Table 1, isolates Kpn-991 and Kpn-1001 were resistant to CZA and susceptible to imipenem, meropenem, meropenem/vaborbac tam, and imipenem/relebactam.Isolate Kpn-1001 was also susceptible to ertapenem, while Kpn-991 was resistant to this agent.Following standard procedure, the isolates were then subjected to carbapenemase screening by in-house CLSI Carba NP test (9), which yielded a negative report for carbapenemase activity.Surprisingly, the presence of bla KPC was revealed by the in-house real-time quantitative polymerase chain reaction (qPCR) method (10), and production of KPC was further confirmed using a NG-Test CARBA 5 (NG Biotech, Guipry, France).
Plasmid sequence BLAST against NCBI GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi)showed that pKpn_1001_KPC-197 is highly similar to two plasmids harbor ing bla KPC-2 , pKpQIL-UK (GenBank: KY798507.1),and pKpUC43 (GenBank: CP115931.1),from two K. pneumoniae isolates collected in the United Kingdom and in the United States, respectively.Likewise, pKpn_991_KPC-31 is very similar to three plasmids that harbor bla KPC-3 , pNJST258C2 (GenBank: CP006919.1),pMu1413 (GenBank: CP096817.1),and pECAZ161_KPC (GenBank: CP019010.1)(Fig. 1).These plasmids were recovered from two K. pneumoniae (pNJST258C2 and pMu1413) and one Escherichia coli isolate from the United States.Further conjugation assays using E. coli J53 as the recipient strain demonstrated that the plasmid containing the bla KPC gene was transferable from Kpn-1001 but not Kpn-991, as confirmed by qPCR performed on transconjugant TC1001.Consistent with the successful transfer and expression of bla KPC , TC1001 displayed an MIC of 32 mg/L for CZA, remaining susceptible to imipenem and meropenem (Table 1).The novel KPC-197 harbors an A177E substitution, a deletion of two amino acids on the Ω-loop (del_168-169_EL), and an insertion of two amino acids in position 274 (Ins_274_DS) (Fig. 2A and C).The deletion of the EL residues at position 167 was previously reported in KPC-66 (amino acid sequence identity, 98.98%) and KPC-92 (98.63%) among others, and the Ins_274_DS has been previously reported in KPC-82 (98.64%) (8,12).Comprehensive biochemical characterization of this novel KPC-197 is currently being undertaken.The sequencing data have been deposited in GenBank database under BioProject number PRJNA1001578 and accession number OR633287 for KPC-179 variant.
Numerous variants of KPC conferring resistance to CZA have been described among clinical isolates worldwide (8).The first CZA-resistant isolate was reported in 2015 in the United States.This K. pneumoniae harbored porin mutations with increased bla KPC-3 expression (13).The first report in South America occurred in 2020 with the identification of three CZA-resistant K. pneumoniae isolates ST11 harboring KPC-8 in Argentina (14).Recently, KPC-113 and KPC-114 were identified from K. pneumoniae isolates belonging to ST11 and ST16 in Brazil (15).KPC-31 has been reported in Italy, the United States, and Argentina (14)(15)(16).As also shown here, this KPC variant confers resistance to CZA at the expense of the carbapenemase activity (7,16).Interestingly, Faccone et al. identified a KPC-31 in a CZA-resistant Pseudomonas aeruginosa ST235, which had been exposed to CZA (17).Indeed, previous studies have shown that prolonged exposure to CZA selects CZA-resistant isolates producing KPC variants ( 18) Colombia is a country endemic for KPC, where the co-circulation of KPC-2 and KPC-3 in Enterobacterales has been well-documented (19)(20)(21).A variety of K. pneumoniae sequence types has been associated with the dissemination of bla KPC-2 ; simultaneously, isolates belonging to the "high risk clone" CG258 have been found to be the main drivers of bla KPC-3 spread (19).Of note, ST45, which one of the isolates described in this study belonged to, has been reported in other studies carrying bla KPC-3 and bla NDM-1 in Colombia (21,22).As far as mobile genetic elements, the most common plasmid replicons in bla KPC -harboring isolates are IncFIB(K), IncFII(K), ColRNAI, IncR, and IncFII.Although Tn4401 transposon remains the most frequent element within most isolates, a study by Saavedra et al. reported a variety of other mobile genetic elements carrying bla KPC (21).
In this context, CZA was a long-awaited addition to the armamentarium against carbapenem-resistant Gram-negatives.CZA therapy was approved by the Colombian health regulatory authorities in 2019.A previous surveillance study conducted on strains collected before the introduction of CZA in Colombia found a 5.7% of resistance to this combination, exclusively due to the production of MBLs in Enterobacterales (23,24).Therefore, this is the first report of CZA resistance in Enterobacterales due to the production of CZA-resistant KPC variants in Colombia.
The two isolates producing KPC variants recovered in our study exhibited resistance to CZA; conversely, they also exhibited susceptibility to imipenem and meropenem, and to the newer β-lactam/β-lactam inhibitor combinations meropenem/vaborbactam and imipenem/relebactam.A trade-off between CZA resistance and decreased carbapene mase activity is often observed among these KPC variants (8).On one hand, this feature makes detection of these KPC variants challenging.As the Carba NP test detects the in vitro hydrolysis of imipenem by a bacterial lysate using the pH indicator phenol red (10), KPC variants with impaired imipenem hydrolysis will not be detected.Thus, molecular tests as PCR or immunoassays such as NG-Test CARBA 5 are needed to track their dissemination.On the other hand, this feature might offer an opportunity for patients infected by CZA-resistant KPC-producing Enterobacterales to be treated with imipenem/relebactam and meropenem/vaborbactam (25).Nevertheless, a marked overproduction of KPC associated with impairment of major porins may led to devel opment of cross-resistance to ceftazidime/avibactam, meropenem/vaborbactam, and imipenem/relebactam in KPC-producing Klebsiella pneumoniae isolates (26,27).
In conclusion, the detection of K. pneumoniae isolates resistant to CZA due to the production of KPC-3 variants in two different cities in Colombia is worrisome.Moreover, it warrants thorough surveillance studies with accurate identification of KPC variants conferring CZA resistance.If the findings described here are only the "tip of the iceberg" and the CZA resistance in the country is more widespread than previously known, the use of this antibiotic as an alternative therapeutic could be severely compromised.

FIG 2 (
FIG 2 (A).Multiple sequence alignment of the amino acid sequence of representative KPC variants and the KPC-197 found in isolate Kpn_1001.Regions of interest for development of CZA resistance, the Ω-loop, loop 237-243, and the loop 266-275 helix are highlighted in red, orange, and yellow, respectively.The active site Ser70 as well as the SDN loop are highlighted in purple.(B) Crystal structure of KPC-2 (PDB 2OV5) with the CZA resistance hot spots highlighted as in (A).(C) Overlay of KPC-2 (light cyan) with a model of the new KPC-197 found in Kpn_1001 generated by SWISS-MODEL (https://swissmodel.expasy.org/)shown in pink.The arrows point to the predicted structural changes in the Ω-and 266-275 loops of the novel KPC-197 variant.

TABLE 2
Genetic characterization of strains K. pneumoniae 991 and 1001